Overexpression of GEM-Cbl-b suppresses FcϵRI-mediated activation of MAP kinases and cytokine gene transcription in RBL-2H3 cells. (A) Analysis of FcϵRI-mediated cytokine gene transcription. Control cells and cells overexpressing GEM-Cbl-b and GEM-Cbl-b (C373A) were cultured overnight with anti-DNP IgE and then stimulated with 30 ng/mL antigen DNP-BSA or 10 ng/mL PMA plus 0.5 μM A23187 (P/I). After the indicated times, total RNA was extracted and hybridized with the ³²P-labeled RNA probes. The protected double-stranded RNA was separated by the urea gel and analyzed by the autoradiography. Similar results were obtained when the other cloned lines were examined. The results were representative of 10 experiments. (B-E) Control cells and cells overexpressing GEM-Cbl-b and GEM-Cbl-b (C373A) were primed with anti-DNP IgE and stimulated with 30 ng/mL antigen DNP-BSA for the indicated times. Total cell lysates or detergent-soluble cell lysates (1-3 × 10⁵ cell equivalents per lane) were separated by SDS-PAGE and analyzed by the immunoblotting with antiphospho-SAPK/JNK (pJNK) and anti-JNK (B), antiphospho-p44/42 ERK (pERK) and anti-p44/42 ERK (C), and antiphospho-p38 MAP kinase (p-p38) and anti-p38 MAP kinase antibodies (D), antiphospho-IKKα/β (pIKKα/β) and anti-IKK antibodies (E), respectively. The numbers at the bottom of the figures are the normalized densitometric analysis of JNK, ERK, p38, and IKK phosphorylation. Similar results were obtained when the other cloned lines were examined. The results were representative of at least 3 experiments (B-E).